The proposed research is a dissection of the targeting and action of the RNA chaperone CYT-19 protein as it facilitates folding of a group I RNA. Although RNA chaperones were proposed long ago, the first demonstration that any protein functions naturally to chaperone RNA folding was in 2002, when Lambowitz and colleagues showed that the Neurospora crassa CYT-19 protein functions by accelerating folding of several group I introns. These introns also require the splicing factor CYT-18, additionally suggesting that CYT-19 may be targeted to RNAs by CYT-18. CYT-19 is a DExD/H-box protein, which are present in all organisms and involved in virtually every process that involves structured RNA, including essential processes like ribosome biogenesis and pre-mRNA splicing, as well the replication of viruses including HCV. DExD/H-box proteins are generally thought to use ATP binding and hydrolysis to mediate structural rearrangements of RNA, facilitating folding to a functional structure or transitions between functional structures. However, little is known about their mechanisms of action or about what governs their specificity for RNA or RNA-protein substrates, largely because of the complexity of many of their targets. The system above provides a unique opportunity for biochemical dissection because it is sufficiently simple that it can be readily manipulated, yet sufficiently complete that it captures a physiological action of a DExD/H-box protein. However, folding of Neurospora group I introns is not well characterized and the best-studied intron populates multiple misfolded forms, inhibiting a deep biochemical dissection. This proposal therefore focuses instead on the group I RNA from Tetrahymena and its derivative that binds CYT-18. The Tetrahymena RNA is extensively characterized and folds to a discrete misfolded form whose re-folding to the native state is also accelerated by CYT-19. Specific aims are to 1) Use kinetics approaches to probe the molecular action of CYT-19 in re-folding the Tetrahymena ribozyme, comparing the reaction to nonspecific unwinding of duplex RNA and testing specific models for the mechanism of ribozyme re-folding; 2) Examine determinants and mechanisms of specificity conferred by the CYT-18 protein; 3) Use sedimentation equilibrium to probe the self-association behavior of CYT-19 in solution and bound to RNA, then use this information to probe the multimeric state of CYT-19 as it acts; 4) Explore an unexpected activity of CYT-19, dissociation of an oligonucleotide substrate from the ribozyme. These experiments are intended to provide novel and fundamental understanding of how a DExD/H-box protein acts as an RNA chaperone and how it is targeted. Results here are expected to guide models for the action and targeting of DExD/H box proteins involved in all aspects of RNA metabolism and function.